Age-related changes of cell death pathways in rat extraocular muscle

Abstract

Changes in the structure and function of aging non-locomotor muscles remains understudied, despite their importance for daily living. Extraocular muscles (EOMs) have a high incidence of age-related mitochondrial defects possibly because of the metabolic stress resulting from their fast and constant activity. Apoptosis and autophagy (type I and II cell death, respectively) are linked to defects in mitochondrial function and contribute to sarcopenia in hind limb muscles. Therefore, we hypothesized that apoptosis and autophagy are altered with age in the EOMs. Muscles from 6-, 18-, and 30-month-old male Fisher 344-Brown Norway rats were used to investigate type I cell death, caspase-3, -8, -9, and -12 activity, and type II cell death. Apoptosis, as measured by TUNEL positive nuclei, and mono- and oligo-nucleosomal content, did not change with age. Similarly, caspase-3, -8, -9, and -12 activity was not affected by aging. By contrast, autophagy, as estimated by gene expression of Atg5 and Atg7, and protein abundance of LC3 was lower in EOMs of aged rats. Based on these data, we suggest that the decrease in autophagy with age leads to the accumulation of damaged organelles, particularly mitochondria, which results in the decrease in function observed in EOM with age.

Figure 1. Autophagy is present in EOM at all ages studied

Ultrastructural analyses by means of electron microscopy of ultrathin sections from EOMs of rats at 6- (left), 18-(middle) and 30-(right) months of age. The arrows indicate autophagic vacuoles: 6-mo (left) autophagocytic degradation with sarcolemmal preservation, bold arrows indicate lipid droplets. 18-mo (middle) top arrow indicates a degradative autophagic vacuole that has fused with a lysosome (AVd/Ly) and 30-mo (right) A late autophagic vacuole (AVd). (Scale bars=2μm for 6-mo, 0.2μm for 18 and 30-mo).

Figure 2. Autophagy markers decrease with age in EOM

Results are -fold changes of the corresponding mRNA in EOM muscles at 18-, and 30-mo compared to 6-mo. * significantly different from 6-mo, p<0.05.

Figure 3. LC3 decreases with age in EOM

A. Muscle cross sections stained for LC3 display a positive staining of autophagic vacuoles in EOM from 6- (left) and 18- (middle) but not in 30- (right) month old rats. Scale bare = 25μm. B. Western blot of LC3 I (top band) and II (botom band) showing decrease protein abundance at 30-mo. C. Quantificaton of optical density of LC3 western blot. * different from 6-mo, ** different from 18-mo(p<0.05).

Figure 4. Mitochondrial size in EOM decreases with age

Quantificaton of mitochondrial width in EOM. Width is significantly decreased in 18- and 30- mo compared to 6-mo rats. * different from 6-mo (p<0.05). Values are means ± SEM.